Long wavelength long lifetime luminophores

ABSTRACT

A new approach is described to making luminophores which display long emission wavelengths, long decay times, and high quantum yields. These luminophores are covalently linked or otherwise closely associated pairs with a long lifetime resonance energy transfer (RET) donor e.g., a ruthenium (Ru) metal-ligand complex, and a long wavelength acceptor, e.g., Texas Red. The donor and acceptor can be covalently linked by, e.g., poly-proline spacers. The long lifetime donor results in a long lived component in the acceptor decay which is due to RET. The quantum yield of the luminophores approaches that of the higher quantum yield acceptor, rather than the lower quantum yield typical of metal-ligand complexes. The emission maxima and decay time of such tandem luminophores can be readily adjusted by selection of the donor, acceptor and distance between them. Luminophores with these useful spectral properties can also be donor-acceptor pairs brought into close proximity by some biochemical association reaction. Luminophores with long wavelength emission and long lifetimes have numerous applications in biophysics, clinical diagnostics, DNA analysis and drug discovery.

This work was supported by NIH grant NCRR-08119 and GM 35154; thegovernment may have rights in this invention.

BACKGROUND OF THE INVENTION

In fluorescence spectroscopy the information available from anexperiment is related to the spectral properties of the fluorophore. Forexample, the anisotropy decay of fluorophores which display nanosecond(ns) decay times can be used to measure motions on the ns timescale. Agood number of fluorophores have become available which display red ornear infrared (NIR) emission [1-2]. Such probes are widely used in thebiochemical and medical applications of fluorescence, including proteinlabeling, chromatography, measurements in blood, noninvasive medicaltesting, DNA sequencing and analysis and in vivo measurements [3-13].Many of the red/NIR fluorophores display high extinction coefficientsand good quantum yields, both of which indicate the absorportion andemisson electronic transitions are strongly-allowed. Consequently, thedecay times of the red/NIR probes are typically below 4 ns and oftenbelow 1 ns, as is predicted by theory [14]. These fluorophores typicallydisplay small Stokes' shifts, and scattered light is most difficult toeliminate at wavelengths close to the excitation wavelength.

If slower motions on the μs timescale are of interest then it isnecessary to use fluorophores which display μs decay times. Furthermore,intracellular fluorophores which require UV excitation result in abackground of undesired emission due to the intrinsic fluorescence ofcells and tissues. This autofluorescence from biological samples ismostly on the ns timescale and its intensity decreases at longerexcitation and emission wavelengths. The signal-to-background ratiocannot be significantly improved by gated detection after the excitationpulse. Hence, the signals detected with red or NIR probes can beaffected by scattered light and/or sample autofluorescence.

For these reasons, for example, there is a need for infraredfluorophores which display long excitation and long emission wavelengthsand long decay times and preferably high quantum yields.

SUMMARY OF THE INVENTION

This invention relates to red/NIR luminophores which display both longdecay times and high quantum yields and preferably large Stokes shifts.

In one aspect, this invention provides a method of providing a probewhich emits luminophore radiation in the range of a wavelength λ₁ ofabout 400 nm to about 1200 nm with a high quantum yield Q₁ and a halflife greater than about 25 ns, comprising placing a donor molecule D,which per se emits radiation of a wavelength less than λ₁ with a quantumyield substantially lower than Q₁, in close association with an acceptormolecule A sufficient for resonant energy transfer from D to A, as aresult of which D resonantly transfers energy to A and A emits saidluminophore radiation.

In another aspect this invention provides a luminophore comprising adonor portion (D) in close association with an acceptor portion (A)sufficient for resonant energy transfer from D to A, wherein uponexcitation by external electromagnetic radiation of a wavelength shorterthan λ₁, said luminophore emits luminophore radiation of a wavelengthlonger than λ₁, which is in the range of about 400 to about 1200 nm withan emission lifetime τ₁ and a quantum yield Q₁,

wherein when D is not in said close association with A, it absorbsradiation of a wavelength λ₂ shorter than λ₁ and thereafter emitsradiation with a quantum yield Q₂ less than about 0.2,

wherein when said donor portion D is in said close association with Aand is excited by electromagnetic radiation of wavelength shorter thanλ₁, it resonantly transfers energy to said acceptor portion A which thenresonantly emits said luminophore radiation, and wherein said quantumyield Q₁ is substantially greater than Q₂.

For example, this invention provides a compound of the formulaD-L-A

-   -   wherein D is a donor metal ligand complex having a quantum yield        less than about 0.2 for emission in the wavelength range of        greater than about 400 nm;    -   A is an acceptor of energy resonantly transferred from D which        is then emitted in the wavelength range of about 400 to about        1200 nm; and    -   L is a spacer of a length effective for resonant energy transfer        between D and A.

In another aspect, this invention provides a chemical compound markedwith a covalently bonded detectable label which is a compound above andprovides the corresponding methods of labeling compounds and identifyingthe latter in a mixture of compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

Various other features and attendant advantages of the present inventionwill be more fully appreciated as the same becomes better understoodwhen considered in conjunction with the accompanying drawings, in whichlike reference characters designate the same or similar parts throughoutthe several views, and wherein:

FIG. 1 shows the chemical structure of a Ru MLC covalently linked toTexas Red (D-A); wherein the donor-alone control has the sulfhydrylgroup blocked with iodoacetamide and the acceptor alone was the peptidewithout the MLC group.

FIG. 2 shows a Jablonski diagram for an irreversible excited stateprocess.

FIG. 3 shows the effect of energy transfer efficiency on the totalquantum yield.

FIG. 4 shows the simulated time-dependent decays of the donor andacceptor, each alone and in a D-A pair; for these simulations τ_(D)⁰=1000 ns and τ_(A) ⁰=10 ns.

FIG. 5 shows the emission spectra of the Ru-(pro)₆ donor (D) the TRacceptor (A) and the covalently linked pair (D-A) in aqueous buffer.

FIG. 6 shows the absorption (top) and excitation spectra (bottom) ofRu-pro)₆(D), TR(A), and Ru-(pro)₆-TR(D-A) in aqueous buffer.

FIG. 7 shows the ratio of the absorption spectra (top) and emissionspectra (bottom) of the D-A pair divided by that of the acceptor inaqueous buffer.

FIG. 8 shows the frequency domain intensity decays of the donor alone(D), acceptor alone (A) and of the covalently linked D-pro₆-A pair inaqueous buffer (top) and in propylene glycol (bottom).

FIG. 9 shows the reconstructed time-domain intensity decays of the donoralone (D), acceptor alone (A) and the covalently linked pair (D-A) inwater (top) and in propylene glycol (bottom); the solid line t_(DA) isfor D-pro₆-A and the dashed-dotted (-•-•-) line t_(DA) is for D-pro₈-A.

FIG. 10 shows the frequency domain intensity decays of the donor alone(D), acceptor alone (A) and of the covalently linked D-pro₈-cys-A pairin the aqueous buffer (top) and in propylene glycol (bottom).

FIG. 11. A potential long wavelength, long lifetime luminophore based ona long lifetime donor (D) and a short lifetime acceptor (A).

FIG. 12. Intuitive description of resonance energy transfer from a highquantum yield donor (Q_(D)=1.0, top) and a low quantum yield donor(Q_(D)=0.1, bottom). For both panels ε_(A)/ε_(D)=0.1. For the lowquantum yield donor RET results in an increase in the overall quantumefficiency of the tandem luminophore.

FIG. 13. Chemical structures of the donors and acceptors used in thisreport.

FIG. 14. Emission spectra of the acridine orange donor [Donor]=5 μMbound to DNA in the presence of the acceptors nile blue (top), TOTO-3(middle), or TO-PRO-3 (bottom). The inserts show the acceptor regionwith the amplitude increased by a factor of 40. The dashed lines showthe emission of acceptor-alone with DNA, but without donor, at thehighest acceptor concentration used in the figure. The donor is presentat 1 donor per 200 base pairs. The number for the donor-acceptor (DA)pair is the number of base pairs for each acceptor.

FIG. 15. Emission spectra of ethidium bromide donor [Donor]=10 μM boundto DNA in the presence of the acceptors nile blue (top), TOTO-3 (middle)or TO-PRO-3 (bottom). The dashed lines show the emission of theacceptor-alone with DNA, but without donor, at the highest used acceptorconcentration. The donor is present at 1 donor per 100 base pairs. Thenumber for the donor-acceptor (DA) pair is the number of base pairs foreach acceptor.

FIG. 16. Emission spectra of Ru-BD([Ru(bpy)₂dppz]²⁺) donor [Donor]=20 μMbound to DNA in the presence of the acceptors nile blue (top), TOTO-3(middle) and TO-PRO-3 (bottom). The dashed lines show the emissionspectra of the acceptor-alone with DNA, but without donor, at thehighest used acceptor concentration. One donor is present per 50 basepairs. The number for the donor-acceptor (DA) pair is the number of basepairs for each acceptor.

FIG. 17. Uncorrected excitation spectra of Ru-BD and TO-PRO-3 bound toDNA (

), and of TO-PRO-3 alone bound to DNA (

). The lower panel shows the ratio of the two excitation spectra.

FIG. 18. Transmission spectra of the emission filters used for measuringthe frequency-domain intensity decays (

). The dashed lines show representative emission spectra of the donoralone (D) and donor plus acceptor (DA) samples.

FIG. 19. Frequency-domain intensity decays of Ru-BD bound to DNA in theabsence and presence of the nile blue acceptor. The solid dots representthe phase or modulation values and the solid lines the bestmulti-exponential fits to the data. In the middle and lower panels thedotted lines represent the donor-alone and acceptor-alone frequencyresponses, respectively.

FIG. 20. Frequency-domain intensity decay of Ru-BD bound to DNA in theabsence and presence of the TOTO-3 acceptor. See legend to FIG. 7.

FIG. 21. Frequency-domain intensity decay of Ru-BD bound to DNA in theabsence and presence of the TO-PRO-3 acceptor. See legend to FIG. 7.

FIG. 22. Time-domain intensity decay of Ru-BD and acceptor complexeswith DNA.

Merely by way of example, the invention is illustrated by the tandemluminophore shown in FIG. 1. This luminophore displays resonance energytransfer (RET) from the exemplary ruthenium metal-ligand complex (MLC)donor shown to the exemplary Texas red (TR) acceptor. The termluminophore is used because emission from these particular MLCs displayboth singlet and triplet character. In no way is this term to limit thisinvention. A metal ligand complex is used as the donor because thetransition from the triplet excited state to the singlet ground state isnot allowed and these molecules display long lifetimes ranging from 100ns to 10 μs [15-17]. Some MLCs are known which display still longerdecay times from 50 to 260 μs [18-20]. Because of the long lifetimes,ease of synthesis, and range of spectral properties, the MLCs have beendeveloped as luminescent probes in physical, analytical and biophysicalchemistry [21-28].

While the MLCs display some favorable spectral properties, otherproperties are less favorable. For example, the MLCs display lowextinction coefficients, typically less than 20,000 M⁻¹ cm⁻¹, e.g., near10,000 M⁻¹ cm⁻¹; which is one reason for the long decay times [14], butwhich results in decreased sensitivity. Additionally, most MLCs displaylow quantum yields which rarely exceed 0.1, and the quantum yields ofthe MLCs with the longest decay times are often smaller [18-20].Finally, the emission spectra are broad, which makes it more difficultto quantify the MLC emission in the presence of autofluorescence becausethe background is also widely distributed across the wavelength scale.Broad emission spectra also result in significant spectral overlap ofthe emission spectra of various MLCs, and an inability to usemeasurements at multiple emission wavelengths to resolve multiplespecies in a macroscopic or microscopic samples.

In the present invention, these limitations of the available MLC andred/NIR probes are overcome. The luminophore of this invention comprisesa MLC which displays a long lifetime and low quantum yield and which is,e.g., covalently linked to a high quantum yield acceptor which typicallyis a short lifetime fluorophore. The luminophore is excited at awavelength where the MLC absorbs, typically near 450 nm for theexemplary ruthenium (Ru) MLCs. The emission therefrom is red shifted tolonger wavelengths by RET to the red/NIR emitting acceptor. Some longwavelength probes have low absorption near 450 nm so that most of theincident light is absorbed by the donor. Much if not most of theacceptor emission is thus due to energy transfer from the MLC.

Following pulsed excitation, the excited state population of the MLCbecomes the only excitation source for the acceptor, which continues toemit as long as MLCs remain in the excited state. Such luminophores canstill display long decay times in the presence of RET. For instance, ifthe MLC donor displays a lifetime of 1 μs in the absence of RET, thelifetime of the luminophore is expected to decrease to 100 ns if the RETefficiency is 90%, e.g., D-A distance being 0.7 Ro (Förster distance). Adecay time of 100 ns is much longer than can be obtained with knownred/NIR probes and 100 ns is longer than most autofluorescence. With a10 μs decay time donor, 90% transfer efficiency will result in a 1 μscomponent in the acceptor decay.

Assuming that the acceptor does not absorb at the donor excitationwavelength (λ_(D) ^(ex)), the acceptor is excited solely by RET from thedonor. Since the acceptor lifetime is short (τ_(D)=1 ns), the acceptorintensity will closely follow the donor intensity. Hence the acceptorwill display the same decay time as the donor and the acceptor decaytime (τ_(AD)) will be near 100 ns. Most acceptors will display someabsorption at the donor excitation wavelength. In this case the acceptoremission will typically display two decay times, a ns component due todirectly excited acceptor, and long decay time near 100 ns due to RETfrom the donor. The long lifetime emission acceptor can be readilyisolated with gated detection, which is readily accomplished with photomultiplier tubes (PMTs) [78-80]. Gated detection is frequently used inimmunoassay based on the lanthanides [81, 82].

An important advantage of such a RET probe (FIG. 11) is an increase inthe effective quantum yield of the long lifetime luminophore. Thiseffect is illustrated in FIG. 12. Suppose the donor and acceptor bothdisplay quantum yields of unity (Q_(D)=Q_(A)=1.0). In this case (top)RET quenches the donor and results in an equivalent increase in theemission intensity of the acceptor. The integrated or total intensity ofthe donor and acceptor remains the same in the presence or absence ofRET.

A surprisingly different result is obtained if the donor displays a lowquantum yield. For example, the commonly used ruthenium MLCs havequantum yields of 0.05 or less. In this case the donor emission withoutRET is much weaker (FIG. 12, lower panel). However, RET to a nearbyacceptor still results in the same increased intensity of the acceptor.More specifically, the transfer efficiency can approach unity eventhough the donor quantum yield is low. A favorable result of efficientRET from the donor is that the wavelength integrated intensity of theD-A pair can be almost 20-fold larger than that of the donor or acceptoralone. More specifically, for 100% transfer efficiency, the overallquantum yield becomes the quantum yield of the acceptor. Theseconsiderations suggest that tandem RET probes based on MLC donors can beused to create long lifetime probes, with red-NIR emission, with theadded advantage of an increased effective quantum yield. Additionally,the modular design of these probes allows practical and rationaladjustment of the spectra properties including the excitation andemission wavelengths and the decay times.

Luminophores of this invention are typified in FIG. 11, which shows along lifetime donor (D) which is covalently linked to an acceptor (A),with spectral properties such that resonance energy transfer occurs withmoderate to high efficiency. In this case the D-to-A distance is assumedto be 0.7 R₀, where R₀ is the Förster distance,. This separation resultsin approximately 90% transfer. The donor is preferably a luminescenttransition metal-ligand complex (MLC). Many such MLCs are known, andthey can display a wide range of absorption and emission wavelengths andlong decay times ranging from 100 ns to 10 μs [15-16]. In recent yearsthese complexes have been developed for use as luminescent probes [21,22] for studies of protein dynamics, immunoassays and chemical sensing[23-28].

The theory and application of RET have been described in numerousreviews [31-33]. (The following discussion of theory is in no wayintended to be limiting.) Discussed here are those aspects of RET neededto demonstrate the occurrence of a RET enhanced quantum yield and theappearance of a long lifetime component in the acceptor decay. The rateof energy transfer for a donor to an acceptor is given by

$\begin{matrix}{k_{T} = {\frac{1}{\tau_{D}^{0}}\left( \frac{R_{0}}{r} \right)^{6}}} & (1)\end{matrix}$where τ_(D) ⁰ is the donor lifetime in the absence of acceptor, r is thedonor-to-acceptor distance, and R₀ is the Förster distance at which RETis 50% efficient. The value of R₀ can be accurately calculated from thespectral properties of the donor and acceptor.

Consider the donor-acceptor pair FIG. 1. Assume the donor has a lifetimeτ_(D) ⁰=1 μs and the acceptor a lifetime of τ_(A) ⁰=1 ns when directlyexcited. The efficiency of energy transfer is given by the ratio of thetransfer rate to the total rate of donor deactivation, which is thereciprocal of the lifetime. Hence the transfer efficiency (E) from thedonor is given by

$\begin{matrix}{E = {\frac{k_{T}}{k_{T} + \Gamma_{D}} = \frac{k_{T}}{\lambda_{D} + k_{D} + k_{T}}}} & (2)\end{matrix}$where Γ_(D)=(τ_(D) ⁰)⁻¹=(λ_(D)+k_(D))⁻¹ is the decay rate of the donorin the absence of acceptor, and λ_(D) and k_(D) are the radiative andnon-radiative decay rates, respectively (FIG. 2). The transferefficiency (E) can be determined experimentally from the relativeintensities of the donor in the absence (F_(D)) and presence (F_(DA)) ofacceptor

$\begin{matrix}{E = {1 - \frac{F_{DA}}{F_{D}}}} & (3)\end{matrix}$The transfer efficiency can also be determined from the donor decaytimes in the absence (τ_(D) ⁰) or presence (τ_(D)) of acceptors

$\begin{matrix}{E = {1 - \frac{\tau_{D}}{\tau_{D}^{0}}}} & (4)\end{matrix}$This expression is only valid when the donor decay is a singleexponential. The decay time of the donor in the presence of acceptor isgiven byτ_(D)=1/(k _(T)+Γ_(D))  (5)which is the reciprocal of the sum of the deactivation rates of thedonor.

The possibility of using rapid RET to improve the system quantum yieldwith low quantum yield donors can be seen from the equations whichdescribe the donor (F_(D)) or acceptor (F_(A)) intensities. In thekinetic scheme of FIG. 2, the intensity of the donor and acceptor isproportional to the amount of light absorbed or the extinctioncoefficient (ε_(D) and ε_(A)) and the fraction of the absorbed lightwhich is emitted. Hence in the absence of RET

$\begin{matrix}{F_{D}^{\circ} = {\frac{\lambda_{D}ɛ_{D}}{\lambda_{D} + k_{D}} = {{Q_{D}^{0}ɛ_{D}} = {\tau_{D}^{0}\lambda_{D}ɛ_{D}}}}} & (6) \\{F_{A}^{\circ} = {\frac{\lambda_{A}ɛ_{A}}{\lambda_{A} + k_{A}} = {{Q_{A}^{0}ɛ_{A}} = {\tau_{A}^{0}\lambda_{A}ɛ_{A}}}}} & (7)\end{matrix}$

where ε_(A) and ε_(D) are the extinction coefficients at the wavelengthused to excite the donor. The lifetimes of the unquenched donor and thedirectly excited acceptor are given by (τ_(D) ⁰)⁻¹=λ_(D)+k_(D) and(τ_(A) ⁰)⁻¹=λ_(A)+k_(A). The quantum yields of the donors or acceptorsin the absence of energy transfer are given by the ratio of the emissiverates (λ_(D) or λ_(A)) to the sum of the rate process which depopulatesthe excited state (λ_(D)+k_(D)) or (λ_(A)+k_(A)). There is usually someacceptor emission even in the absence of RET due to direct absorption(excitation) of the acceptor resulting from the non-zero value of ε_(A).For clarity the proportionality constant is dropped which should be onthe right side of each equations 6 and 7.

In the absence of RET the total intensity (F_(T) ^(o)) of the donor(F_(D) ^(o)) and acceptor (F_(A) ^(o)) is that due to direct excitationof both species

$\begin{matrix}{F_{T}^{\circ} = {{F_{D}^{\circ} + F_{A}^{\circ}} = {{\frac{\lambda_{D}ɛ_{D}}{\lambda_{D} + k_{D}} + \frac{\lambda_{A}ɛ_{A}}{\lambda_{A} + k_{A}}} = {{Q_{D}^{0}ɛ_{D}} + {Q_{A}^{0}ɛ_{A}}}}}} & (8)\end{matrix}$where F_(T) ^(o) is the total emission in the absence of transfer. Nowassume RET occurs with a rate k_(T). The intensities of the donor andacceptor are given by

$\begin{matrix}{F_{D} = {\frac{\lambda_{D}ɛ_{D}}{\lambda_{D} + k_{D} + k_{T}} = {Q_{D}ɛ_{D}}}} & (9) \\{F_{A} = {\frac{\lambda_{A}ɛ_{A}}{\lambda_{A} + k_{A}} + {\frac{k_{T}ɛ_{D}}{\lambda_{D} + k_{D} + k_{T}} \cdot \frac{\lambda_{A}}{\lambda_{A} + k_{A}}}}} & (10)\end{matrix}$

The intensity or quantum yield of the donorQ_(D)=λ_(D)/(λ_(D)+k_(D)+k_(T)) is decreased by an additional rate k_(T)which depopulates the donor (eq. 9). The intensity of the acceptor isincreased by the transfer rate k_(T). The transfer efficiency termE=k_(T)/(λ_(D)+k_(D)+k_(T)) in eq. 10 can be understood as the fractionof absorbed photons absorbed by the donor which are transferred to theacceptor. These transferred photons are emitted with a quantum yieldQ_(A)=λ_(A)/(λ_(A)+k_(A)). The energy received from the donor is emittedwith the quantum yield of the acceptor. The combined emission intensityof the donor and acceptor is given byF _(T) =F _(D) +F _(A) =Q _(D)ε_(D) +Q _(A) ^(o)(ε_(A) +Eε _(D))=Q _(D)⁰ε_(D)(1−E)+Q _(A) ⁰(ε_(A) +Eε _(D))  (11)

It is instructive to consider the limits of very slow (k_(T)→0 and E→0)and very fast (k_(T)→∞) energy transfer. In the limit of no energytransfer the total intensity becomes equal to that of a mixture of twonon-interacting fluorophores (eq. 8). In the limit of rapid transfer(k_(T)→∞ and E→1) the total intensity becomes

$\begin{matrix}{F_{T} = {\frac{\lambda_{A}\left( {ɛ_{A} + ɛ_{D}} \right)}{\lambda_{A} + k_{A}} = {Q_{A}\left( {ɛ_{A} + ɛ_{D}} \right)}}} & (12)\end{matrix}$This is an important result which indicates the total intensity isproportional to the sum of the extinction coefficients and to thequantum yield of the acceptor. This occurs because the energy transfercan occur with an efficiency of one even if the donor quantum yield islow. If the rate of energy transfer is fast and if the acceptor absorbsweakly the excitation wavelength (ε_(A)<<ε_(D)) then

$\begin{matrix}{F_{T} = {\frac{\lambda_{A}ɛ_{D}}{\lambda_{A} + k_{A}} = {Q_{A}ɛ_{D}}}} & (13)\end{matrix}$This equation shows that with rapid energy transfer and no directlyexcited acceptor the acceptor emission intensity is proportional to theamount of light absorbed by the donor and the quantum yield of theacceptor. The donor-acceptor pair becomes essential to a new fluorophorewith an extinction coefficient E_(D) and a quantum yield Q_(A).

It is informative to consider the time-dependent decays of the donor,acceptor and the total emission. These expressions are similar to thoseknown for an excited state reaction [34-37]. Here, the reverse transferrate from A to D is zero (FIG. 2). Additionally, since both donor andacceptor are present all times, there is some direct excitation of theacceptor in addition to the acceptor which is excited by RET from thedonor. The time-dependent changes in the donor and acceptor populationsare given by

$\begin{matrix}{\frac{\mathbb{d}\lbrack D\rbrack}{\mathbb{d}t} = {{- {\left( {\Gamma_{D} + k_{T}} \right)\lbrack D\rbrack}} + {ɛ_{D}{L(t)}}}} & (14) \\{\frac{\mathbb{d}\lbrack A\rbrack}{\mathbb{d}t} = {{- {\Gamma_{A}\lbrack A\rbrack}} + {k_{T}\lbrack D\rbrack} + {ɛ_{A}{L(t)}}}} & (15)\end{matrix}$

where L(t) is the excitation function. The square brackets are taken toindicate the excited state population of each species. Thetime-dependent decays of the donor and acceptor are given byI _(o)(t)=N _(D) ⁰ exp[−Γ_(D) +K _(T))t]  (16)I _(A)(t)=A exp[−Γ_(D) +k _(T))t]−(N ^(o) _(A) −A) exp[Γ_(A) t]  (17)

where N_(D) ⁰ and N_(A) ⁰ are the number of excited donors and acceptormolecules at t=0. The pre-exponential factors in eqs. 16 and 17 areproportional to ε_(D)L(t) and ε_(A)L(t), respectively, but not shown.The factor A

$\begin{matrix}{A = {\frac{N_{D}^{0}k_{T}}{\Gamma_{A} - \Gamma_{D} - k_{T}} = \frac{{- N_{D}^{0}}k_{T}}{\Gamma_{D} - \Gamma_{A} + k_{T}}}} & (18)\end{matrix}$

depends on the efficiency by which the acceptor is pumped by the donor.According to equation 16, the donor decay I_(A) (t) is the usual decayrate of a donor with a transfer rate k_(T). The acceptor decay containsa component with the lifetime of the acceptor τ_(A)

0 and a component with the lifetime of the quenched donor τ_(D).

Suppose the acceptor decay is very rapid, that is, the directly excitedacceptor displays a short lifetime, τ_(A)

⁰ 6 0 or Γ_(A) is very large. Then the acceptor decay becomesI _(A)(t)=A exp[−(Γ_(A) ⁻ +k _(T))t].  (19)This result shows that in the limit of a short acceptor lifetime theacceptor emission resulting from energy transfer displays the samelifetime as the quenched donor. A similar result is shown if one assumesτ_(D)>>τ_(A) or Γ_(A)>>Γ_(D). In this case the rightmost term inequation 17 decays rapidly to zero, relative to the donor decay, and theacceptor decay resulting from RET displays the same decay time as thedonor. If there are no initially excited acceptors, N_(A) ⁰=0, equal andopposite pre-exponential factors are obtained and the acceptor decaysaccording toI _(A)(t)=A exp[−(Γ_(D) +k _(T))t]−A exp[Γ_(A) t]  (20)

Moreover, the inventor's publication, Lakowicz et al., AnalyticalBiochemistry 288, 62-75 (2001) is entirely incorporated by referenceherein.

In one aspect, this invention thus involves the increase of theeffective quantum yield of a luminophore by rapid RET in long lifetimeMLC components having low quantum yields. Such an increase in effectivequantum yield has not previously been important in the biochemical usesof RET [29-30 and 38-46] because most organic donors have good quantumyields. The increased effective quantum yield of the donor has not beenimportant for RET with, e.g., the lanthanides because transfer from theorganic chelates to the lanthanides is efficient, and the shieldedlanthanide donors often display quantum yields near unity [42-46]. (Seealso the enhancement of lanthanide emission when bound to essentialnon-luminescent DNA or nucleotides [47-49]). There are numerous primaryreports and review articles on RET, and the concept of using theacceptor emission to measure the transfer efficiency is not new [38-41].Additionally, Selvin and co-workers have already noted the usefulness ofmeasuring the long lifetime acceptor emission with lanthanide donors toselectively detect D-A pairs [42] and to provide a long decay time forthe acceptor [43, 44]. Donors and acceptors with short decay times havebeen covalently linked for use in DNA sequencing [30, and Ju et al.PNAS, USA, 92, 4347 (1995)] and as high affinity dyes which bindnon-covalently to DNA [45, 46].

The approach of this invention to tandem luminophores can be rationallyand routinely used to obtain the desired spectral properties. RET is ahighly predictable phenomena. The long acceptor decay time can beincreased by a longer spacer. Less spectral overlap of the D and A canbe obtained using shorter wavelength rhenium MLC donors or longerwavelength acceptors.

These tandem luminophores can be prepared in conjugatable forms and usedas a single reagent. This invention can also be applied to themeasurement of protein or DNA association reactions where the donor andacceptor are present in separate molecules and are placed in closeassociation by the interactions of the separate molecules.

The luminophores of this invention can be used as labels fully analogousto prior art labels, e.g., those discussed in the references citedherein, e.g., by conventional covalent linking to desired molecules tobe detected, e.g., nucleic acid proteins, cells, etc., probes basedthereon etc.

Thus, this invention involves donor molecules/portions, D, typicallyhaving low quantum-yields less than about 0.2 or even lower, e.g., about0.1 or about 0.01-0.2, 0.1-0.2, etc. Such donor molecules are wellknown. Typically they are metal ligand complexes, of transition metals(e.g., atomic numbers 21-30, 39-48 and 72-80); those of the lanthanides(e.g. atomic numbers 57-71, 81-83) are also possible, but thesetypically have high quantum yields. A wide variety of well knowndonor-type metal ligand complexes are well known. See references 15-27.See, as well, Demas et al., Coordination Chemistry Reviews 211 (2001)317-351; Stufkens et al., Coordination Chemistry Reviews 177 (1998)127-179. Typically, but not in a limiting way, these are of the di-iminee.g., bipyridyl type. Most preferred are the transition metal complexes,especially those of renium, ruthenium, osmium and iridium. Such Dmolecules are well known as having low quantum yields and having broademission spectra at relatively long wavelengths, as mentioned above.Their emission life times are also relatively long as also mentionedabove.

The acceptor molecules/portions are also per se well known in the field.Typically, these are dye molecules such as Texas Red. Albumin 633 or670, CY5, fluorescein dyes, polymethine dyes, cyanine dyes, squariliumdyes, croconium dyes, merocyanine dyes, oxonol dyes, and many others.See e.g., WO 98/22146; and topics in Fluorescence Spectroscopy, Vol. 4:Probe Design and Chemical Sensing, ed. Joseph R. Lakowicz, Plenum Press,N.Y., 1994, Chapter 6, R. B. Thompson, pp. 151-182, and Chapter 7,Guillermo A. Casay, et al., pp. 183-222. These acceptor molecules areknown as having high quantum yields per se and as emitting in relativelylong wavelength regions with long lived decay times.

This invention provides a combination of molecules or closely associatedcomponent species involving both D and A molecules/portions e.g.,covalently linked to one another or in close association with each othersuch that the spacing of the two molecules, in all cases, is effectivefor resonant energy transfer from the donor to the acceptor. This may beachieved not only by covalent linking but also by use of conventionalbiological association reactions, e.g., nucleic acid hybridizationbetween two nucleic acid molecules (DNA, RNA, etc.), one bonded to thedonor and the other bonded to the acceptor. Such association can also beachieved by other similar specifically interacting molecules, e.g.,protein/nucleic acid, antibody/antigen, receptor/ligand, etc. Details ofthe linking of the donor and/or acceptor molecules/portions to any suchmolecules are fully conventional.

Where a D/A molecule is to be employed, the D portion is linked to the Aportion by a spacer or linker molecule, L. The nature of the spacer isnon-critical, the effective parameter being the distance between D and Aand the covalently linked combination. Thus, any of the well knownspacer molecules can be employed, e.g., polyalkylene moieties, polyaminoacid moieties (e.g., polyproline moieties of the examples), maleimidomoieties, isothiocyanate moieties, esters, ethers, secondary andtertiary amines, amides, the structures cited below, etc.

See any of the well known prior art linker-related disclosures in thisregard. In general, the closer D and A are spaced from each other thefaster and more efficient will be the resonant energy transfer, e.g., ascan be seen from the examples. Determination of an optimal distance anda corresponding spacer is fully routine as can be seen from theliterature cited herein. Typically, spacings are desired which willachieve transfer efficiencies about 10%-90%, e.g., 20-80%, 30-70%,40-60%, efficiencies around 50% typically being satisfactory. If thetransfer efficiency is too high, then the decay times achieved will betoo short.

As can be seen, by routine selection of the D-moiety, A-moiety andspacer distance, “designer” probes can be achieved in accordance withthis invention. See, e.g., Stufkens et al., above, e.g., pp.171-174;Chen et al., J. Am. Chem. Soc. 2000, 122, 657-660. Typically, theresultant long wavelength emission will be in the range of 400-1200 nm,e.g., 450-1200, 550-1000 nm and more typically 600-900 nm. Decay lifetimes (half lives) will typically be greater than 25 ns, typically 25ns-100 μ, more typically 50 ns-10 μs, and most typically 50 ns-2 μs.Luminophores of this invention having a desired emission wavelength andlifetime can be prepared in accordance with well known considerationsand the guidance provided by this specification. Selection of the A andD moieties appropriate for a desired emission wavelength range can bemade using conventional considerations e.g., as discussed in references15-27, e.g., by suitable routine selection of metal and ligandcombinations. Modification of the spacing length between D and A willsimilarly routinely be achievable by appropriate selection of chemicallinking moieties, to achieve a resultant desired transfer efficiency andlife time.

The production of the D and A compounds according to the invention canbe carried out by conventional modification of the substances, whichcontain functionalities that can be coupled (e.g., carboxyl, amino, andhydroxyl groups), according to processes well known to one skilled inthe art.

The production of the adducts according to the invention is carried outby reaction of the dye with a metal ligand complex or ligand complex(followed by metallation) according to methods that are well known inthe literature. The dyes and complexes must have reactive groups thatcan be coupled in this regard or they must routinely be activatedin-situ or in advance by generation of these groups. With regard, e.g.,to amino- and sulfhydryl groups suitable reactive groups are, forexample, N-hydroxysuccinimidylester,N-hydroxy-succinimidylester-3-sulfate, isothiocyanates, isocyanates,maleimide-, haloacetyl, vinylsulfone groups. The coupling is preferablycarried out in an aqueous medium. In this case, the degree ofconcentration can be routinely controlled by stoichiometry and reactiontime. See e.g., Snyth. Commun. 23 (1993) 3078-94, DE-OS 3912046, CancerImmunol. Immunother. 41 (1995) 257-263, Cancer Research 54 (1994)2643-49.

Thus, as can be seen, this invention provides luminiphor probes emittinglong wavelength radiation with high quantum yield despite theinvolvement of absorbing donors having low quantum yields. As a result,emitter probes are provided at wavelengths to which skin is at leasttranslucent, in which wavelength ranges background autofluorescence andnatural fluorophore emissions are minimized. Such long lifetime emissionis achieved also despite the use of acceptor portions (dyes) per sehaving short life times. This represents another significant advantagesince extant background fluorescence tends to be of significantlyshorter lifetimes than that achieved by the emitters of this invention.

The closely associated D/A pairs of this invention can be usedstraightforwardly in any of the usual probe-based techniques mentionedherein, e.g., including nucleic acid sequencing, hybridization assays,immunoassays, etc. This aspect is fully conventional. See e.g., Ota etal., Nucleic Acid Research, 1998, Vol. 26, No. 3, 735-743; Peterson etat., J. Am-Chem. Soc., 2000, 122, 7837-7838; Paris et al. Nucleic AcidResearch, 1998, Vol. 29, No. 16, 3789-3793; Templeton et al., Clin.Chem. 37/9, 1506-1512 (1991); Weissleder et al., Nature BiotechnologyVol. 17, April 1999, 375-378; Xiav et al., Proc. Natl. Acad Sci.,95,15309-15314, December 1998.

Another application of this invention is for the study of macromolecularassociation reactions, such as protein-protein interactions, DNAhybridization [58-60], fluorescence in-situ hybridization (FISH) [61],or the use of molecular beacons [62, 63]. As an example, suppose it wasnecessary to test for binding of donor-labeled oligonucleotides to amixture of acceptor-labeled oligonucleotides. When using a RuMLC donorand one of the acceptors used in this report, most of the specieslabeled with donor or acceptor alone will display little emission. Incontrast the D-A pairs due to macromolecular association will bebrightly fluorescent. Additionally, the acceptor emission will be longlived. Using time-gated detection brightly fluorescent spots may becomeapparent against background of weakly stained chromatin and/or shortdecay time. These spectral properties will be useful for detection ofoligonucleotide hybridization on DNA arrays [64-65]. Such arrays arebecoming widely used for analysis of gene expression [66-68].

Thus, a generic approach to obtaining an unusual combination of spectralproperties by using an appropriate D-A pairs is provided. This approachcan be used to create D-A pairs which acts as a single luminophore, orthis effect can be used to detect interactions in samples containingspecies labeled with the donor or acceptor. This approach will also beuseful in studies of macromolecular folding as illustrated by the use ofRET to study ribozyme structures [69, 70]. One can also provide longlifetime donors linked to pH, Ca²⁺, or other analyte-sensitivefluorophores [71, 72]. If the analyte sensitive fluorophore displaysdistinct emission spectra with and without bound analyte, then therewill be a long lived component in the emission with the spectralcharacteristics of each form. Finally, the use of the enhanced emission,and inhibition of the enhancement, can be used in macromolecular bindingassays in high throughput screening [73, 74]. There appear to benumerous applications of our approach in biochemical and biomedicalresearch.

ABBREVIATIONS A acceptor D donor D-A donor-acceptor pair MLCmetal-ligand complexes NIR near infrared PMT photomultiplier tube TRTexas Red bpy 2,2′-bipyridine phen 1,10-phenanthroline RET resonanceenergy transfer Ru Ru(bpy)₂(phen-ITC) which has been covalently linkedto a peptide or DNA oligomer

In the foregoing and in the following examples, all temperatures are setforth uncorrected in degrees Celsius; and, unless otherwise indicated,all parts and percentages are by weight.

The entire disclosures of all applications, patents and publications,cited above and below are hereby incorporated by reference.

EXAMPLE 1

Simulations were performed to predict the spectral properties of the D-Apair for typical decay times and quantum yields. For these simulations,eq. 11 was modified to use the normalized extinction coefficient ε′_(D)and ε′_(A)

whereQ _(T) =Q _(D) ⁰ε′_(D)(1−E)+Q _(A) ⁰(ε′_(A) +Eε′ _(D))  (21)ε′_(D)=ε_(D)/(ε_(D)+ε_(A))  (22)andε′_(A)=ε_(A/(ε) _(D)+ε_(A))  (23)FIG. 3 shows the total quantum yield expected for three D-A pairs forvarious transfer efficiencies. The quantum yield of the acceptor wasassumed to be high Q_(A) ⁰=0.9 (top), intermediate Q_(A) ⁰=0.5 (middle)and low Q_(A) ⁰=0.1 (lower panel). Since most acceptors will absorb atthe donor excitation wavelength, we assumed the normalized extinctioncoefficient of the acceptor was ε′_(A)=ε_(A)/(ε_(A)+ε_(D))=0.10. As thetransfer efficiency increases the total quantum yield approaches that ofthe acceptor. If the acceptor quantum yield is low (lower panel), thenenergy transfer decreases the overall quantum yield. Importantly, if thequantum yield of the acceptor is high (upper panel), the overall quantumyield approaches that of the acceptor for high transfer efficiency.

The intensity decays expected for the donor and acceptor in D-A pairsfor various transfer efficiencies (FIG. 4) were also simulated. Theassumed decay times were τ_(D) ⁰=1000 ns and τ_(A) ⁰=10 ns. An importantconclusion from these simulations is that the acceptor can display longdecay times. If the transfer efficiency is 33% (FIG. 4, top panel), theacceptor shows a decay time with τ=667 ns (Table I). The transferefficiency can be as high as 90.9% and the acceptor still display a 91ns decay time. Thus, usefully long decay times can be obtained even withhigh transfer efficiency.

EXAMPLE 2

The practical usefulness of the tandem luminophores of this inventionwere demonstrated using the covalently linked D-A pairs shown in FIG. 1.These D-A pairs can be considered to be the probe or reagent, in thesame manner that linked DNA pairs have been developed for DNA sequencing[29-30]. Alternatively, this unique long lifetime high quantum yieldemission can be the result of protein or nucleic acid associationreactions.

The Texas Red iodoacetamide with a C5 linker was purchased fromMolecular Probes, Inc. The [Ru(bpy)₂ (amino phenanthroline)]²⁺ was agift from Dr. Jonathan Dattelbaum. It was converted into isothiocyanateby treating with 500 μl of thiophosgene in 1 ml acetone for 3 hrs. Boththe solvent and thiophosgene were removed under a stream of nitrogen andthe isothiocynate was used immediately.

The oligo proline peptides with a cysteine at C-terminus weresynthesized at the biopolymer facility of University of Maryland Schoolof Medicine, Baltimore. The crude peptide was purified by RP-HPLC on aC18 column using a 0.1% TFA and 100% acetonitrole containing 0.05% TFA.The molecular weights were confirmed by mass spectroscopy.

The peptides were labeled first with the acceptor. Typically a mMsolution of the peptide in 0.2 M bicarbonate buffer, pH 8.5, was reactedwith a 2-fold excess iodoacetamide for 6 hours. The resulting peptidewas purified from the free probe using a column of Sephadex G-15 runningin 20% DMF solution. The labeled peptide was further purified by HPLC.

To prepare the double labeled peptide the acceptor labeled peptide wasfurther reacted with a five-fold excess Ru isothiocynate in 0.2 Mbicarbonate, pH 9.0 for 6 hours. The peptide was separated from the freeprobe by passing through a Sephadex G-15 column and further purified onHPLC. To prepare the donor-only peptide, the sulphydryl group was firstblocked with a five-fold excess iodoacetic acid at pH 8.5 for 1 hr andto same reaction mixture a five-fold excess of the isothiocyanate wasadded, the pH was adjusted to 9 and allowed to react for 6 hours. Thefree dye was separated on a Sephadex G-15 column and the donor-labeledpeptide was purified by HPLC. The purified peptides were lyophilized andstored as water solutions at 4° C.

The steady-state measurements were done in an aqueous 5 mM hepes, 100 mMNaCl, pH 8. The measurements in propylene glycol were without bufferwith the propylene glycol at least 98%, the remainder being water. Forthe steady-state measurements the peptide concentrations were less than2 μM and about 10 μM for the time-resolved measurements. An aqueoussolution of rhodamine B with a lifetime of 1.68 ns was used as thereference. The frequency-domain lifetime measurements were done on a SLMinstrument with a LED emitting at 450 nm as a light source. The emissionwas observed through a 630/40 nm bandpass filter.

The emission spectra of Ru-(pro)₆-cys-TR (FIG. 1), referred to as the(pro)₆ D-A pair was examined. As a control for the donor-alone (D), thestructure shown in FIG. 1 was used with the sulfhydryl group blockedwith iodoacetamide. For the acceptor (A), the structure shown in FIG. 1was used without the covalently linked donors. Emission spectra of thesethree compounds are shown in FIG. 5. These spectra were obtained usingthe same molar concentrations of D, A and D-A. The overall intensity ofthe D-A pair is about 5-fold larger than the sum of the donor andacceptor alone. This result demonstrates that a tandem luminophore witha low quantum yield donor can display a higher quantum yield than eitherspecies alone.

FIG. 6 shows the absorption and excitation spectra of D, A and D-A. Theabsorption spectra of D-A was found to be essentially identical to thesum of the D-alone and A-alone absorption spectra (top). Contrastingresults were found for the excitation spectra (FIG. 6, bottom). In thiscase the intensity of the long wavelength emission with excitation at450 nm is about 6-fold greater than that of the directly excitedacceptor and about 20-fold larger than the donor alone. This result alsodemonstrates the role of energy transfer in increasing the effectivequantum yield of the donor.

The enhanced emission demonstrated in FIGS. 5 and 6 is determined by therelative extinction coefficients of the donor and acceptor at theexcitation wavelength. The ratio of the donor to the absorption spectrais shown in the top panel of FIG. 7. This ratio displays a maximum near6 at 450 nm, which is near the peak of the donor absorption and theminimum of the acceptor absorption. The ratio of the excitation spectrashows the same trend, with a maximum near 450 nm (FIG. 7, bottom). Theseresults demonstrate that the enhancement at the acceptor emission isdetermined by the ratio of the light absorbed by each species.

Data also showed that the enhanced red emission could be obtained withusefully long decay times. This is an important consideration because ifthe donor and acceptor are too close, or the rate of transfer is toofast, then the donor decay time will be shortened towards the ns valuecharacteristic of the directly excited acceptor. The frequency-domainintensity decay of D, A and D-A are shown in water (FIG. 8, top) and inpropylene glycol (bottom). For ease of understanding, thefrequency-domain data were used to reconstruct the time-dependent decays(FIG. 9). In the absence of acceptor, the donor-alone displays a mostlysingle exponential decay with a decay time of 515 ns (top). The donordecay time is longer in propylene glycol (bottom), near 820 ns. Thedecay time of the directly excited acceptor is much shorter and near 4ns in either solvent.

The D-A pair measured at the acceptor emission wavelength displays amore complex intensity decay, as can be seen from the frequencyresponses for D-pro₆-A (FIG. 8) or D-pro₈-A (FIG. 9). The acceptor inD-pro₈-A displays a longer decay time as seen from the shift to lowerfrequency of D-pro₈-A as compared to D-pro₆-A The reconstructedintensity decays are shown in FIG. 10 and the intensity decay parametersare summarized in Table II. For a D-A pair at a single distance, asingle decay time is expected for the donor. The heterogeneous decays ofthe D-A pairs is probably the result of a range of D-to-A distances dueto the flexibility of the linkers between hexaproline and the probes.There are 12 chemical bonds between the last proline and Texas Red. Whenusing an acceptor with a shorter linker, a more mono-exponential decaywill result. Nonetheless, the D-pro₆-A displays a long decay time near22 ns in water and 55 ns in propylene glycol. The components areassigned as due to the acceptors which are being excited by the exciteddonor population. A greater than 10-fold reduction in the donor decaytime due to RET is consistent with the greater than 90% RET efficiencyshown by this D-A pair.

Similar data were collected for the larger D-A pair with the pro₈spacer, D-pro₈-A (Table II). The frequency-domain data are shown in FIG.9 and the time-domain representations are shown in FIG. 10. For thismore widely spaced D-A pair the acceptor shows a decay time of 50 ns inwater and 130 ns in propylene glycol. Hence long decay times exceeding100 ns can be obtained using such tandem luminophores.

TABLE I Expected Lifetimes and Total Quantum Yields for D-A pairs^(a)Acceptor Fluorescence Total Quantum Yield of the Transfer EfficiencyLifetimes System E τ₁ [ns] τ₂ [ns] Q_(T) = Q_(D) + Q_(A) 0 10 — 0.1080.091 10 909 0.180 0.333 10 667 0.372 0.500 10 500 0.504 0.667 10 3330.636 0.833 10 167 0.768 0.909 10 91 0.829 0.950 10 48 0.860 0.980 10 200.884a τ_(D) ⁰=1000 ns, τ_(A) ⁰=10 ns, Q_(D) ⁰=0.02, Q_(A) ⁰=0.90. For thesecalculations we assumed the extinction coefficient of the donor is9-fold larger than that of the acceptor, at the excitation wavelength.

TABLE II Multi-exponential intensity decay analysis for the donor,acceptor and DA pairs shown in Scheme I^(a) Solvent/ Compound Water Qα_(i) ^(b) f_(i) τ_(i) χ_(R) ² D-pro₆ 0.0333 0.099 0.009 41.2 1.45^(c)0.901 0.991 516.7 pro₆-A 0.360 1.0 1.0 4.0 1.26 D-pro₆-A 0.33 0.4690.198 3.5 0.82 0.353 0.304 7.0 0.178 0.458 22.7 D-pro₈-A — 0.784 0.2874.4 0.137 0.252 22.3 0.079 0.461 71.1 Propylene Glycol D-pro-₆ — 0.1250.014 79.4 0.98 0.875 0.986 785 pro₆-A 1.0 1.0 4.1 2.36 D-pro₆-A — 0.8030.363 7.9 0.178 0.245 33.4 0.069 0.392 99.5 0.51 D-pro₈-A — 0.839 0.2255.1 1.3 0.089 0.170 36.3 0.072 0.604 157.8 ^(a)Excitation was at 455 nmusing a blue light emitting diode. The emission was measured at 630 nmwith a 25 nm bandpass. ^(b)The decays were analyzed internally at themulti-exponential model, I(t) = Σ3α_(i) exp(−t/τ_(i)), ƒ₁ =α_(i)τ_(i)/Σα_(j)τ_(j) ^(c)δp = 0.3° and δm = 0.003.

EXAMPLE 3

Materials: CT-DNA, Tris.HCl and EDTA was obtained from Sigma (St. Louis,Mo.). Ru-BD was synthesized by the method described previously [51,52].AO, EB, TOTO-3 and TO-PRO-3 were purchased from Molecular Probes(Eugene, Oreg.) and NB was from Aldrich (Milwaukee, Wis.). All reagentswere used without further purification and water was deionized with aMilli-Q system. To convert CT-DNA into linear fragments comparable inlength to one persistent length, about 5 mg/ml solution of CT-DNA wassonicated approximately 10 min while submerged in an ice bath. Thesonicated DNA solution was centrifuged for 1 hr at 75,000 ×g to removetitanium particles and undissolved DNA. All experiments were undertakenat room temperature in 2 mM Tris.HCl, pH 8.0, containing 0.1 mM EDTA.

Absorption and steady-state fluorescence measurement: AO, EB and Ru-BDserved as donors and NB, TOTO-3 and TO-PRO-3 were used as acceptors.About 5-10 mM stock solutions of AO, Ru-BD and NB were prepared indimethylformamide and about a 10 mM stock solution of EB were made inDMSO. The final DMF concentration in all solutions was less than 1%(v/v). The concentration of DNA was quantified using a molar extinctioncoefficient of 13,300 M⁻¹ cm⁻¹ (expressed as bp) at 260 nm. The DNAconcentration was 1 mM bp while the concentrations of AO, EB and Ru-BDwere 5, 10 and 20 μM, respectively. Concentration of the probes weredetermined using the extinction coefficients in Table III. The highestacceptor concentrations of Ru-BD/NB, Ru-BD/TOTO-3, and Ru-BD/TO PRO-3D-A pairs were 120, 60 and 120 μM, respectively. Because TOTO-3 andTO-PRO-3 were supplied as 1 mM stock solutions in DMSO, the maximumpercentages of DMSO in the Ru-BD/TOTO-3 and Ru-BD/TO-PRO-3 D-A pairswere 6 and 12%(v/v), respectively. In preliminary experiments, we foundthat DMSO increased the steady-state fluorescence intensity of RuBD(data not shown). Hence, we added aliquots of DMSO to obtain 6 and12%(v/v) DMSO in all Ru-BD/TOTO-3 and Ru-BD/TO-PRO-3 D-A pairs,respectively, to equalize the effect of DMSO. UV-visible absorptionspectra were measured with a Hewlett-Packard 8453 diode arrayspectrophotometer with ±1 nm resolution. Steady-state fluorescencemeasurements were carried out using an Aminco SLM AB2 spectrofluorometer(Spectronic Instruments, Inc., IL) under magic angle conditions. Theexcitation wavelengths of AO, EB and RuBD were 470, 518 and 440 nm,respectively.

Frequency-domain fluorescence measurements: Measurements were performedusing the instruments described previously [75] and modified with a dataacquisition card from ISS, Inc. (Urbana, Ill.) [76]. The excitationsource was a blue LED LNG992CFBW (Panasonic, Japan) with luminousintensity of 1,500 mcd, and an LED driver LDX-3412 (ILX Lightwave,Boseman, Mo.) provided 30 mA of current at frequencies from 1 to 9.3MHz. A 450RD55 interference filter (Omega Optical, Inc., Brattleboro,Vt.) and a 4-96 color glass filter (Corning Glass Work, Corning, N.Y.)were used to isolate the excitation wavelength. Rhodamine B in water wasutilized as a lifetime standard. The transmission curves of the filtersfor isolating the emission from the donor, D-A pairs, and acceptors areshown below (FIG. 18).

Steady State Spectra

DNA with non-covalently bound donors and acceptors was used to test thepossibility of creating long lifetime luminophores with high quantumyields. Three donors, acridine orange (AO), ethidium bromide (EB) and[Ru(bpy)₂dppz]²⁺ (Ru-BD) were chosen. These structures are shown in FIG.13. When bound in DNA the quantum yields decrease in this respectiveorder (Table III). Acceptors, were nile blue (NB), TOTO-3 and TO-PRO-3(FIG. 13), which display increasing quantum yields in the listed order.Dyes non-covalently bound to DNA were used because this approach allowedus to select donors and acceptors with various quantum yields, withoutthe need for chemical synthesis. Also, this approach allowed us toadjust the concentrations of donors and acceptors to observe trends inthe spectra. Based on the theory described above, the largest overallincrease in the total emission of the tandem luminophore was expected tooccur with RET between the lowest quantum yield donor (Ru-BD) and thehighest quantum yield acceptor (TO-PRO-3).

FIG. 14 shows the emission spectra of AO bound to DNA with increasingamounts of acceptor. With the high quantum yield AO donor the NBacceptor emission is almost undetectable (FIG. 14, top insert). Thequantum yield of the TOTO-3 acceptor is higher than that of NB, and thequantum yield of TO-PRO-3 is higher still. The acceptor emission becomesmore easily detectable as the acceptor quantum yields increase. In eachcase the observed acceptor emission is due to RET from the donor. Nosignificant acceptor emission was found for the acceptors bound to DNAin the absence of donor (dashed lines). An interesting aspect of FIG. 14is that RET from a high quantum yield donor (AD) to a low quantum yieldacceptor (NB) decreases the total emission from the donor and acceptor.

FIGS. 15 and 16 show emission spectra with the same acceptors, but withEB and Ru-BD as the donors. Examination of these spectra shows that theenhancement of the acceptor emission is larger for Ru-BD than for EB.Also, the largest enhancements are seen for TO-PRO-3, the acceptor withthe highest quantum yield (FIG. 16, lower panel). In this case theacceptor emission is increased many-fold by energy transfer from theRu-BD donor. Also, the emission from the D-A system is considerablylarger than that of the donor alone bound to DNA, or the acceptor alonebound to DNA (dashed line). This effect is the opposite of that foundfor the AO/NB D-A pair. In this case the weakly fluorescent NB receivedmost of the energy by RET, but still emits with its own low quantumyield. For the Ru-BD/TO-PRO-3 D-A pair the strongly fluorescent TO-PRO-3receives most of the energy absorbed by the donor, in spite of the lowintrinsic quantum yield of the donor.

In the absence of energy transfer the intensity of the acceptor isproportional to ε_(A)Q_(A), where ε_(A) refers to the extinctioncoefficient of the acceptor at the donor excitation wavelength. Iftransfer is 100% effective the intensity of the acceptor is proportionalto (ε_(A)+ε_(D))/ε_(A). According to Table III this ratio is near 4.Examination of FIG. 16 (lowest panel) indicates that the acceptorenhancement is greater than 4, surprisingly. To further this effect theexcitation spectra of the D-A pair, and the acceptor alone, when boundto DNA were examined. On the same relative scale the acceptor alonedisplays essentially no emission upon excitation at 450 nm (FIG. 17).The lower panel shows the ratio of these excitation spectra, whichbecomes close to 40 at 450 nm. This ratio is larger than expected fromthe extinction coefficients listed in Table III. It appears thatexcitation of TO-PRO-3 near 450 nm results in less emission thanpredicted by its absorption spectrum. This effect could be due to thepresence of non-flourescent absorbing impurities, or absorption ofnon-fluorescent conformers of TO-PRO-3 at 450 nm. It is known that thisclass of dyes display weak fluorescence in water or when there istorsional motions about the central methine bridge [77]. Irrespective ofthe origin of this low intensity, the acceptor enhancement seen in FIG.16 is consistent with the excitation spectrum for this D-A pair.

Time-Resolved Decays

Frequency-domain intensity decays were measured through filters selectedto isolate the desired emission wavelengths (FIG. 18). Observation at610 nm results in selective observation of the donor emission, andobservation at 670 or 700 nm selects the acceptor emission.

FIGS. 19-21 show the frequency-domain data for three D-A pairs. In thesedata Ru-BP is always the donor. The acceptor is NB, TOTO-3 or TO-PRO-3,respectively. In the absence of acceptors, the mean Ru-BD lifetime isnear 100 ns (Table IV). The Ru-BD lifetime is only moderately decreasedby the acceptor. For instance, for any of the acceptors, a ratio of 0.03acceptors per base pair results in a mean donor lifetime is near 70 ns.This was initially surprising given the 2-fold or larger quenching ofthe Ru-BD intensity by these acceptor concentrations. However, thisdifference in intensity and lifetime quenching can be explained as dueto a range of D-to-A distances in the labeled DNA. More specifically,most of the acceptor emission results from the more closely spaced D-Apairs. In contrast, the observed donor emission in the presence ofacceptors is increased by the higher intensities of those donors mostdistant from acceptors, which are also the donors with the longerlifetimes.

The lower panels of FIGS. 19-21 shows the frequency response observedfor the longer wavelength regions dominated by the acceptor emission. Ineach case the mean decay times are near 30 ns for observation at theacceptor emission wavelengths. While the frequency responses aremulti-exponential, visually obvious contributions from the directlyexcited acceptors with their 0.3 to 2.3 ns lifetimes were not found. Theapparent acceptor lifetimes are shorter than the apparent donorlifetimes because the acceptor emission is enriched for the shorterdistances D-A pairs which have a shorter donor lifetime.

It is informative to examine the intensity decays in the time-domainreconstructed from the frequency-domain data (FIG. 22). The decays ofthe directly excited acceptors are short, and emission from the directlyexcited acceptors will not be observed if the detection is off-gated forthe first 10-20 ns following the excitation pulse. The donor decays,even in the presence of acceptors, are long lived. Also, following abrief transition period out to 10-40 ns, the acceptor decay rates arecomparable to that of the quenched donors. This long lived emission fromthe donors can be used for biophysical or analytical purposes.

An important conclusion from these experiments is that the apparentacceptor decays are adequately long for off-gating of theautofluorescence from biological samples. Hence the use of MLC-acceptorpairs provides an opportunity to obtain luminophores which display longlifetimes, high quantum yields, and long emission wavelengths.

By consideration of the well known characteristics of Förster transfer,one can predict that suitable designed D-A pairs will display even morefavorable properties. For instance, the acceptor decay times for the DNAbound probes were shorter than the donor decay times. This effect is dueto a range of donor-to-acceptor distances for the probes randomly boundto DNA. It is well known that unique D-to-A distances can be obtainedwith polyproline spacers [53] or with double-stranded DNA as the spacer[54-55]. In such cases the donor decay times will decrease in proportionto the transfer efficiency, and the acceptor decay times will be similarto the donor decay times. The results for a donor and acceptor separatedby a single distance are expected to be comparable to that shown in FIG.11, where a 1 μs decay time donor, with 90% transfer efficiency, resultsin a luminophore with a 100 ns lifetime. Since metal-ligand complexesare known with decay times as long as 42 μs [56-57], one can predict 4μs decay time luminophores with 90% transfer.

Another advantage of these RET probes is that the emission spectra ofred and NIR fluorophores are typically narrow on the wavelength scale,whereas the emission spectra of the MLCs are broad. Sinceautofluorescence from biological samples is typically broaderdistributed broadly on the wavelength scale, the concentration of theemission into a narrow spectral range by the acceptor will improvedetectability of these luminophores.

TABLE III Quantum Yields (Q), Decay Times (τ) and Molar ExtinctionCoefficients (ε/λ_(max)) of Fluorophores in DNA Donor/ ε/λ_(ex)ε/λ_(max) Probe Acceptor Q^(a) τ (ns) (M⁻¹cm⁻¹/nm) (M⁻¹cm⁻¹/nm) AO Donor0.392 5.0 23,300/470  53,000/500 EB Donor 0.219 21.9 5,200/518 5,200/518 RuBD Donor 0.008 84.0 13,000/440  13,000/440 NB Acceptor0.004 0.32 1,180/440 42,900/656 TOTO-3 Acceptor 0.06 2.3 2,240/440154,000/642  TO-PRO-3 Acceptor 0.11 1.8   200/440 102,000/642  ^(a)Thefollowing compounds were used as quantum yield references: in the caseof AO, 3-aminofluoranthene in DMSO (Q = 0.32); EB in methanol (Q = 0.06)for EB; 4-dicyanomethylene-2-methyl-6-(p-dimethylaminostyryl)4H-pyran inmethanol (Q = 0.38) in the case of RuBD and NB; and fluorescein in 0.1 MNaOH (Q = 0.92) for TOTO-3 and TO-PRO-3. ^(b)Mean lifetime calculatedusing τ = Σ f_(i) τ_(i), where f_(i) is the fractional steady statecontribution of each component to the total emission. ^(c)From MolecularProbes, Inc.

TABLE IV Multi-exponential intensity decay anlayses of the Ru-BD donorand acceptors bound to calf thymus DNA. Donor/Acceptor n^(a) <τ>^(b)α_(i) f_(i) τ_(i) χ_(R) ² Ru-BD/NB^(c) Ru-BD 2 84 0.36 0.13 24 0.91 0.640.87 93 NB 1 0.32 1.00 1.00 0.32 0.85 DA Obs. 610 nm 2 75 0.50 0.15 161.40 0.50 0.85 86 DA Obs. 700 nm. 3 30 0.95 0.51 1.9 0.90 0.04 0.21 220.01 0.28 87 Ru-BD/TOTO-3^(c) Ru-BD 2 98 0.42 0.18 33 0.99 0.58 0.82 111TOTO-3 1 2.3 1.00 1.00 2.3 1.30 DA Obs. 610 nm 2 73 0.79 0.19 5.8 1.070.21 0.81 90 DA Obs. 670 nm. 3 39 0.83 0.41 5.6 1.02 0.12 0.24 23 0.050.35 88 Ru-BD/TO-PRO-3^(c) Ru-BD 2 114 0.42 0.17 38 1.02 0.58 0.83 130TO-PRO-3 1 1.8 1.00 1.00 1.8 1.02 DA Obs. 610 nm 2 83 0.62 0.19 14 0.810.38 0.81 99 DA Obs. 670 nm. 3 24 0.83 0.48 5.1 1.05 0.15 0.33 19 0.020.19 78 ^(a)Number of decay times in the multi-exponential fit. ^(b)<τ>= Σ τ_(i) f_(i) where f_(i) is the steady state contribution of eachcomponent. ^(c)All acceptor concentrations are 0.03 bp⁻¹

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples. Also, the preceding specific embodiments are to be construedas merely illustrative, and not limitative of the remainder of thedisclosure in any way whatsoever.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

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1. A luminophore comprising a donor portion (D) in close associationwith an acceptor portion (A) sufficient for resonant energy transfertherebetween, wherein upon excitation by external electromagneticradiation of a wavelength shorter than λ₁, said luminophore emitsluminophore radiation in the range of about 450 to about 1200 nm of awavelength longer than λ₁, with an emission lifetime t₁ and a quantumyield Q₁, wherein when D is not in said close association with A, itabsorbs radiation of a wavelength λ₂ shorter than λ₁ and thereafteremits radiation with a quantum yield Q₂ less than about 0.2, whereinwhen said donor portion is in said close association with A and isexcited by electromagnetic radiation of wavelength shorter than λ₁, itresonantly transfers energy to said acceptor portion A which thenresonantly emits radiation of a wavelength longer than λ₁ with saidemission lifetime t₁ and quantum yield Q₁, which is substantiallygreater than Q₂, wherein said luminophore is a chemical compound,wherein D is covalently linked to A, and has the formula: